Complete genome sequence of a carlavirus identified in grapevine (Vitis sp) in Greece.

Using high-throughput sequencing, we identified a novel carlavirus sequence in a 28-year-old 'Kotsifali' grapevine sample collected in Heraklion (Crete, Greece). Using RT-PCR and 5'/3' RACE together with Sanger sequencing, the complete genome sequence of 8299 nt was confirmed and found to contain five open reading frames (ORFs) but to lack an ORF6, which is present in some members of the genus Carlavirus. The novel sequence is most similar to those of two carlaviruses infecting caper, and taking into account the ICTV nomenclature, we propose the name "grapevine carlavirus 1" for this new virus.

Genetic evidence for the involvement of Dicer-like 2 and 4 as well as Argonaute 2 in the Nicotiana benthamiana response against Pelargonium line pattern virus.

In plants, RNA silencing functions as a potent antiviral mechanism. Virus-derived double-stranded RNAs (dsRNAs) trigger this mechanism, being cleaved by Dicer-like (DCL) enzymes into virus small RNAs (vsRNAs). These vsRNAs guide sequence-specific RNA degradation upon their incorporation into an RNA-induced silencing complex (RISC) that contains a slicer of the Argonaute (AGO) family. Host RNA dependent-RNA polymerases, particularly RDR6, strengthen antiviral silencing by generating more dsRNA templates from RISC-cleavage products that, in turn, are converted into secondary vsRNAs by DCLs. Previous work showed that Pelargonium line pattern virus (PLPV) is a very efficient inducer and target of RNA silencing as PLPV-infected Nicotiana benthamiana plants accumulate extraordinarily high amounts of vsRNAs that, strikingly, are independent of RDR6 activity. Several scenarios may explain these observations including a major contribution of dicing versus slicing for defence against PLPV, as the dicing step would not be affected by the RNA silencing suppressor encoded by the virus, a protein that acts via vsRNA sequestration. Taking advantage of the availability of lines of N. benthamiana with DCL or AGO2 functions impaired, here we have tried to get further insights into the components of the silencing machinery that are involved in anti-PLPV-silencing. Results have shown that DCL4 and, to lesser extent, DCL2 contribute to restrict viral infection. Interestingly, AGO2 apparently makes even a higher contribution in the defence against PLPV, extending the number of viruses that are affected by this particular slicer. The data support that both dicing and slicing activities participate in the host race against PLPV.

Viral Detection: Past, Present, and Future.

Viruses are essentially composed of a nucleic acid (segmented or not, DNA, or RNA) and a protein coat. Despite their simplicity, these small pathogens are responsible for significant economic and humanitarian losses that have had dramatic consequences in the course of human history. Since their discovery, scientists have developed different strategies to efficiently detect viruses, using all possible viral features. Viruses shape, proteins, and nucleic acid are used in viral detection. In this review, the development of these techniques, especially for plant and mammalian viruses, their strengths and weaknesses as well as the latest cutting-edge technologies that may be playing important roles in the years to come are described.

Cucurbit chlorotic yellows virus p22 is a suppressor of local RNA silencing.

RNA silencing is a major antiviral mechanism in plants, which is counteracted by virus-encoded proteins with silencing suppression activity. ORFs encoding putative silencing suppressor proteins that share no structural or sequence homology have been identified in the genomes of four criniviruses. In this study, we investigated the RNA silencing suppression activity of several proteins encoded by the RNA1 (RdRp, p22) and RNA2 (CP, CPm and p26) of cucurbit chlorotic yellows virus (CCYV) using co-agroinfiltration assays on Nicotiana benthamiana plants. Our results indicate that p22 is a suppressor of local RNA silencing that does not interfere with cell-to-cell movement of the RNA silencing signal or with systemic silencing. Furthermore, comparisons of the suppression activity of CCYV p22 with that of two other well-known crinivirus suppressors (CYSDV p25 and ToCV p22) revealed that CCYV p22 is a weaker suppressor of local RNA silencing than the other two proteins. Finally, a comparative sequence analysis of the p22 genes of seven Greek CCYV isolates was performed, revealing a high level of conservation. Taken together, our research advances our knowledge about plant-virus interactions of criniviruses, an emergent group of pathogens that threatens global agriculture.

Combined Activity of DCL2 and DCL3 Is Crucial in the Defense against Potato Spindle Tuber Viroid.

Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response.

Dicer-Like 4 Is Involved in Restricting the Systemic Movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.

Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Ros1 systemic accumulation dramatically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.

Spotlight on Plant Bromodomain Proteins.

Bromodomain-containing proteins (BRD-proteins) are the "readers" of histone lysine acetylation, translating chromatin state into gene expression. They act alone or as components of larger complexes and exhibit diverse functions to regulate gene expression; they participate in chromatin remodeling complexes, mediate histone modifications, serve as scaffolds to recruit transcriptional regulators or act themselves as transcriptional co-activators or repressors. Human BRD-proteins have been extensively studied and have gained interest as potential drug targets for various diseases, whereas in plants, this group of proteins is still not well investigated. In this review, we aimed to concentrate scientific knowledge on these chromatin "readers" with a focus on Arabidopsis. We organized plant BRD-proteins into groups based on their functions and domain architecture and summarized the published work regarding their interactions, activity and diverse functions. Overall, it seems that plant BRD-proteins are indispensable components and fine-tuners of the complex network plants have built to regulate development, flowering, hormone signaling and response to various biotic or abiotic stresses. This work will facilitate the understanding of their roles in plants and highlight BRD-proteins with yet undiscovered functions.

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation.

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection.

MEK5/ERK5/mef2: a novel signaling pathway affected by hepatitis C virus non-enveloped capsid-like particles.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The viral particle is composed of a capsid containing the genome, surrounded by E1 and E2 proteins, however different forms of viral particles have been observed including non-enveloped particles. Previous reports have proposed that hepatitis C non-enveloped capsid-like particles (HCVne) enter cells of hepatic origin via clathrin-mediated endocytosis, during which different signaling events occur. In this report we show that HCVne particles are capable of inducing the recently discovered ERK5 pathway, in a dose dependent way. The ERK5 pathway can be activated by growth factors and other extracellular signals. This specific activation occurs through a well characterized upstream kinase, MEK5, and is capable of inducing gene regulation of mef2. In contrast, when HCV core structural and NS5A non-structural proteins were expressed endogenously no activation of this pathway was detected. These cell signaling events could be of critical importance and might give clues for the elucidation of cellular manifestations associated with HCV infection.

Modulation of IL-2 expression after uptake of hepatitis C virus non-enveloped capsid-like particles: the role of p38 kinase.

Hepatitis C virus (HCV) has been shown to actively replicate in cells of the immune system, altering both their function and cytokine expression. Naked nucleocapsids have been reported in the serum of infected patients. We investigated interference of recombinant non-enveloped capsid-like particles with signaling pathways in T cells. HCV non-enveloped particles (HCVne) internalization was verified in Jurkat and Hut 78 T cells, as well as primary human peripheral blood and intrahepatic mononuclear cells. HCVne uptake leads to activation of the MAPKs-p38 signaling pathway. Using specific phosphoantibodies, signaling pathways inhibitors, and chemical agents, it was demonstrated that p38 activation in T cells correlated with IL-2 transcriptional activation and was accompanied by a parallel increase of IL-2 cytokine secretion. c-fos and egr-1, two transcription factors, essential for IL-2 promoter activity, were also found to be elevated. We propose that HCVne uptake by T lymphocytes results in increased MAPKs-p38 activity and IL-2 expression, thus altering the host immune response.

Detection of Viroid RNA and vd-siRNA in N. benthamiana Plants: Northern Blot Analyses for Viroid and vd-siRNAs.

Viroids are considered the most minimalistic group of pathogens. Despite their presumed inability to encode for proteins, viroids induce several diseases in plants of primary economic importance. The production of viroid derived siRNAs (vd-siRNAs) of 21-24 nt, accompanies viroid infections in plants and results from the activation of the RNA silencing mechanism and specifically the function of Dicer endonucleases. A comprehensive set of experiments for the study and thorough analysis of viroid-infected plants has been developed. Here we present a detailed experimental plan including optimized protocols for plant infection by agroinfiltration, RNA extraction, and northern blot hybridization for the detection of both viroid genomic RNA and vd-siRNAs.

Revisiting the Non-Coding Nature of Pospiviroids.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Endocytosis of hepatitis C virus non-enveloped capsid-like particles induces MAPK-ERK1/2 signaling events.

Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients. The HCV core particle serves as a protective capsid shell for the viral genome and recombinant in vitro assembled HCV core particles induce strong specific immunity. We investigated the post-binding mechanism of recombinant core particle uptake and its intracellular fate. In hepatic cells, these particles are internalized, most likely in a clathrin-dependent pathway, reaching early to late endosomes and finally lysosomes. The endocytic acidic milieu is implicated in trafficking process. Using specific phosphoantibodies, signaling pathway inhibitors and chemical agents, ERK(1/2) was found to be activated in a sustained way after endocytosis, followed by downstream immediate early genes (c-fos and egr-1) modulation. We propose that the intriguing properties of cellular internalization of HCV non-enveloped particles can induce specific ERK(1/2)-MAPKs events that could be important in HCV life cycle and pathogenesis of HCV infection.

Molecular dissection of the prototype foamy virus (PFV) RNA 5'-UTR identifies essential elements of a ribosomal shunt.

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5'-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5'-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A' or sORF B, and on the translation event at the corresponding 5'-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs.

Alpha-1 antitrypsin deficiency impairs lung antibacterial immunity in mice.

Alpha-1 antitrypsin (AAT) is a major inhibitor of serine proteases in mammals. Therefore, its deficiency leads to protease-antiprotease imbalance and a risk for developing lung emphysema. Although therapy with human plasma-purified AAT attenuates AAT deficiency-related emphysema, its impact on lung antibacterial immunity is poorly defined. Here, we examined the effect of AAT therapy on lung protective immunity in AAT-deficient (KO) mice challenged with Streptococcus pneumoniae. AAT-KO mice were highly susceptible to S. pneumoniae, as determined by severe lobar pneumonia and early mortality. Mechanistically, we found that neutrophil-derived elastase (NE) degraded the opsonophagocytically important collectins, surfactant protein A (SP-A) and D (SP-D), which was accompanied by significantly impaired lung bacterial clearance in S. pneumoniae-infected AAT-KO mice. Treatment of S. pneumoniae-infected AAT-KO mice with human AAT protected SP-A and SP-D from NE-mediated degradation and corrected the pulmonary pathology observed in these mice. Likewise, treatment with Sivelestat, a specific inhibitor of NE, also protected collectins from degradation and significantly decreased bacterial loads in S. pneumoniae-infected AAT-KO mice. Our findings show that NE is responsible for the degradation of lung SP-A and SP-D in AAT-KO mice affecting lung protective immunity in AAT deficiency.

DCL-suppressed Nicotiana benthamiana plants: valuable tools in research and biotechnology.

RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next-generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.

SERRATE, a miRNA biogenesis factor, affects viroid infection in Nicotiana benthamiana and Nicotiana tabacum.

Viroids are plant infecting, non - coding RNA molecules of economic importance. Potato spindle tuber viroid (PSTVd), the type species of Pospiviroidae family, has been shown to be affected by specific RNA silencing pathways. Dicer like 1 (DCL1), a key player in micro RNA (miRNA) pathway has been previously linked with PSTVd infectivity. In this report we aim to further dissect the interaction between the miRNA pathway and Pospiviroid virulence. We mainly focused on the Zinc-finger protein SERRATE (SE) a co-factor of DCL1 and core component of miRNA pathway. We generated Nicotiana tabacum and Nicotiana benthamiana SE knock-down plants exhibiting considerable miRNA reduction and strong phenotypic abnormalities. PSTVd infection of SE suppressed plants resulted in a significant viroid reduction, especially at the initial infection stages. This positive correlation between SE levels and viroid infectivity underlines its role in PSTVd life cycle and reveals the importance of the miRNA pathway upon viroid infection.

The unexpected function of a highly conserved YXXΦ motif in HCV core protein.

Hepatitis C virus (HCV) is an RNA positive strand virus, member of the Flaviviridae family. The HCV viral particle is composed of a capsid containing the genome, surrounded by an endoplasmic reticulum (ER)-derived lipid bilayer where E1 and E2 are assembled as heterodimers. However, different forms of viral particles have been identified in the serum of HCV-infected patients, including non-enveloped particles. Previous reports have demonstrated that HCV non-enveloped capsid-like particles (HCVne) can be generated by HCV core protein sequence. This sequence possesses a highly conserved ΥΧΧΦ motif and distal di-leucine motifs that confer primary endocytosis signals, enabling HCVne to enter hepatic cells via clathrin-mediated endocytosis. Although HCV core's primary function is to encapsidate the viral genome, it also interacts with a variety of cellular proteins in order to regulate host cell functions such as gene transcription, lipid metabolism, apoptosis and several signaling pathways. In this report, we demonstrate that the YXXΦ motif of HCV core protein is crucial for the architectural integrity of the particulate form of HCVne. Moreover, we show that the YXXΦ motif in the HCV core sequence plays a pivotal role in the signaling events following HCVne clathrin-mediated endocytosis by inducing the AP-2 clathrin adaptor protein, which in turn redirect HCVne trafficking to the lipid droplets (LDs) via the endosomal-lysosomal pathway. HCVne and LDs co-localization affects the HCV life cycle by enhancing viral replication.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection.

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

Infectious long non-coding RNAs.

Long non protein coding RNAs (lncRNAs) constitute a large category of the RNA world, able to regulate different biological processes. In this review we are focusing on infectious lncRNAs, their classification, pathogenesis and impact on the infected organisms. Here they are presented in two separate groups: 'dependent lncRNAs' (comprising satellites RNA, Hepatitis D virus and lncRNAs of viral origin) which need a helper virus and 'independent lncRNAs' (viroids) that can self-replicate. Even though these lncRNA do not encode any protein, their structure and/or sequence comprise all the necessary information to drive specific interactions with host factors and regulate several cellular functions. These new data that have emerged during the last few years concerning lncRNAs modify the way we understand molecular biology's 'central dogma' and give new perspectives for applications and potential therapeutic strategies.